This invention relates to the use of a water soluble reusable macroligand for the purpose of purification and stabilization of trypsin. This novel composition provides improved trypsin purification results.
Besides insulin, beef pancreas contains many other valuable compounds such as trypsin, chymotrypsin, carboxypeptidase, elastase, lipase, etc. Pancreas is rather expensive and the size of the slaughter limits the total pancreas supply. Therefore, one should make the best possible use of this tissue by recovering most of the above-mentioned biological compounds.
The recovery of trypsin from beef pancreas is of industrial interest since this enzyme has been widely used for medical purposes; beer haze removal; and meat tenderization. It is, however, quite difficult to isolate and purify trypsin by conventional purification procedures, such as precipitation by salts or solvents, since pancreas contains two very similar enzymes (in terms of molecular weight, isoelectric pH, etc.): trypsin and chymotrypsin. The difference between trypsin and chymotrypsin can only be distinguished by the catalytic reaction. Trypsin catalyzes the hydrolysis of only the peptide bond in which the carboxyl group is contributed by either a lysine or an arginine residue, regardless of the length or amino acid sequence of the chain. Chymotrypsin, on the other hand, attacks only the peptide bonds in which the carboxyl group is contributed by one of phenylalanine, tryptophan, and tyrosine.
For the last decade, several affinity chromatographic methods based on proteinaceous inhibitors have been developed for trypsin purification (G. Feinstein, Biochim. Biophys. Acta, 214, 244, 1970; G. Feinstein, Fed. Eur. Biochem. Soc. Lett., 7, 353, 1970; N. Robinson, R. Tye, H. Neurath and K. Walsh, Biochemistry, 10, 2743, 1971). The system is composed of a stationary insoluble matrix, such as derivatives of cellulose, polyacrylamide, polystyrene, beaded agarose, etc., to which a ligand molecule (trypsin inhibitors) is covalently bound. Such a procedure, however, is costly, laborious and time consuming since the ligands are expensive, difficult to prepare, susceptible to degradation and only have a limited operating life. Furthermore, this system uses a packed column that can plug and foul and, therefore, cannot be used on whole broths as can other techniques, e.g. two-phase extraction (G. Johanson, J. of Biotech., 3, 11, 1985). As a result, conventional affinity chromatography can only be used for processing a fairly clear liquid solution containing proteins and/or enzymes.
Ultrafiltration, another widely used technique, can be utilized to separate solutes from one another or solutes from solvents. Besides its ease of scale-up, ultrafiltration possesses a high productivity and it can be operated as batch or continuous systems. There are, however, disadvantages to ultrafiltration because of its low resolution. In practice, a resolution of ten-fold difference in molecular weight is about the best that can be expected.
There is a recent breakthrough in the isolation and purification of biomolecules such as proteins, enzymes and hormones. The technique has been defined as affinity-ultrafiltration where a specific ligand is chemically bound to a macromolecular water soluble polymer (B. Mattiason and M. Ramstorp, Annals of N.Y. Academy of Sciences, 413, 307, 1983). By using a suitable ultrafiltration membrane the desired product will be retained since it formed a complex with the ligandbound macromolecules. The product is then eluted from the polymer under suitable conditions. In this example, concanavalin-A was purified using heat-killed cells of Saccharomyces cerevisiae as the affinity absorbent and D-glucose as the eluant. Further, Adamski-Medda et al (J. Membrane Science, 9, 337, 1981) utilized dextran-p-aminobenzamidine as a macroligand to separate trypsin and chymotrypsin. The results indicated a low degree of binding specificity. In fact, in the presence of the macroligand the filtrate contained 65% of chymotrypsin and 24% of trypsin. Choe et al (Biotech. Letters, 3, 163, 1986) also attempted to separate trypsin from chymotrypsin using soybean trypsin inhibitor as well as p-aminobenzamidine attached to dextran. The results were not very encouraging either, since only 55% of trypsin input was recovered at a purity of 81%. It was almost apparent through the two proceeding studies that dextran exhibited some non-specific affinity toward chymotrypsin as well as trypsin.
The process described in this application is an improved method over the prior art for it provides a higher enzyme to ligand binding ratio, less fouling to the membrane occurs and the binding is more specific. For example, in a batchwise procedure, the ligand described in this application could purify trypsin from a trypsin-chymotrypsin mixture with 90% yield and a purity of 98%.
U.S. Pat. No. 4,350,760 (J-C. Nicolas et al, Sept. 21, 1982) teaches a method for selective separation of at least one protein having an affinity for a ligand, from other substances, some of them having the same affinity for said ligand but also having a high molecular weight, higher than that of the protein to be isolated. The method consists of filtering the solution containing the proteins on a gel excluding the substances with the higher molecular weight. The gel is coupled with a ligand having affinity for the substance to be purified. The present application differs from the patented process in that the substances to be separated have the same molecular weight but have different affinity to the ligand.
Schneider et al (Annals of N.Y. Academy of Sciences, 369, 257, 1981) developed an affinity precipitation technique for the isolation and purification of trypsin from bovine pancreas. In this reference, a water soluble polymer (polyacrylamide) bearing a ligand group (benzamidine) and a precipitation group (benzoic acid) is described which permits a quantitative precipitation of the affinity polymer. In this technique, the polymer is added directly to a crude extract under conditions favoring the binding of the desired protein. The polymer is then precipitated and the supernatant is removed. The protein of interest is then eluted from the polymer under suitable conditions and the polymer can be recycled. Obviously, a procedure using acidic pH for the precipitation of the polymer and for the elution of the bound protein cannot be used for all enzymes, as many are unstable in acidic media. In addition, this method can only be used in a batchwise procedure, whereas the present application also teaches a method for the continuous purification of trypsin.